ABSTRACT Among inhibitors of Gram+ DNA polymerases IIIC and IIIE, several compounds are active against multiple strains of the anaerobic Gram+ bacterium Clostridium difficile (Cdiff). The compounds appear to be selective for Cdiff compared with other Gram+ anaerobes and aerobes, and a lead compound - 2-(3,4-dichlorobenzyl)- 7-(5-morpholinylpentyl)guanine or 359E - is active orally in protecting hamsters from lethal Cdiff infection. The compounds of interest are poorly absorbed orally and too weak to be developed for systemic use against Gram+ aerobe infections. Compound 359E and analogs are tertiary amines, readily form water soluble salts, and are highly effective against Cdiff in vitro and in vivo. Given the increasing prevalence of Clostridium difficile-associated diarrhea (CDAD), including that from highly virulent, toxin-overproducing and/or antibiotic- resistant strains, the need for new and selective antibacterials to treat this disease is growing. Our phase II results strongly suggest that development of the lead compound or an alternative as an oral treatment for Cdiff diarrhea in human patients will result in a novel, first in class drug to treat this emerging infectious disease. The specific aims of the competing renewal of this project are focused on preclinical development of 359E or a closely related lead compound (LC). The aims are to: 1, scale up and begin process development for 359E;2, determine the mechanism of action, selectivity, anticlostridial spectrum, and resistance development of 359E;3, determine oral safety, anti-clostridial efficacy, and absorption of 359E in the hamster;4, synthesize and screen analogs of 359E as backup LC compounds, and designate a candidate for development (CD). Once this has occurred, IND-enabling studies will commence. These include: 5, in vitro ADME studies;6, preclincal analytical, toxicology and toxicokinetic studies;7, safety pharmacology and genotoxicity studies;8, cGMP production of the CD. Once all preclinical studies have been completed, aim 9 will encompass preparation an IND application for the CD as oral treatment for CDAD. Incidence of CDAD is on the rise in the United States and Europe, and Cdiff is the major identified infectious cause of nosocomial diarrhea in patients to whom antibiotics had been previously administered. Vancomycin and metronidazole are first-line therapy for treatment of CDAD, but there have been reports of treatment failure and CDAD recurrence after treatment with metronidazole, and the Centers for Disease Control and Prevention (CDC) has discouraged vancomycin for treatment of CDAD in hospitals to minimize the risk of vancomycin-resistant enterococci and staphylococci. Various treatments are in clinical trials and preclinical development for Cdiff infections, ranging from direct-acting antibacterials to vaccines and compounds to neutralize Cdiff toxins. The results of our phase II studies indicated the strong likelihood that a DNA polymerase III inhibitor will be an effective and non-toxic oral treatment of CDAD.